The human immunodeficiency virus (HIV) is a serious and growing health threat in virtually every part of the world. It has been estimated that over 22 million people are currently infected worldwide, and it is anticipated that over 40 million people will be infected by the end of this decade.
HIV infection typically leads to acquired immunodeficiency syndrome (AIDS) within 8 to 10 years after infection. Individuals with AIDS are subject to opportunistic infections and cancers, leading to severe illness and, ultimately, death. Although various treatments delaying the progression from HIV infection to AIDS are known, these treatments are of limited effectiveness and generally require the use of pharmaceuticals which have adverse side-effects. Moreover, the effectiveness of different drug treatments varies among individuals and no satisfactory system exists to screen different drug combinations for effectiveness in combating HIV infection in a particular subject.
HIV is a retrovirus, closely related to simian immunodeficiency virus (SIV). At least three variants of HIV, known as HIV-1, HIV-2, and HIV-0, are known. It is believed that HIV-1 is the predominant global form of human HIV infection at the present time. HIV-2 is believed to be common in West Africa, but rarer elsewhere in the world. HIV-2 appears to be less pathogenic that HIV-1. In addition to these three HIV variants, the high natural mutation rate of HIV DNA means that virtually every individual infected with HIV carries a slightly different virus. Differences between HIV isolates complicate efforts to devise effective anti-HIV approaches, including drugs and vaccines.
HIV enters target cells by the binding of gp120 (present on the HIV virion) to cellular receptors, followed by fusion of the viral envelope with the plasma membrane of the target cell. The major cellular receptor for the HIV gp120 is cluster of differentiation factor 4 (CD4). The highest levels of CD4 are generally found on T-helper (Th) cells; thus, the consequences of HIV infection are typically most obvious in the Th cell population. HIV can also infect other cells, including macrophages, monocytes, dendritic cells, Langerhans cells, and microglial cells. HIV-1 has a higher affinity for CD4 than does HIV-2, and it is thought that this may contribute to the greater pathogenicity of HIV-1 compared to HIV-2. HIV-1 also requires a chemokine coreceptor (e.g. CCR5 or CXCR4) to gain entry into susceptible cells.
There is evidence in the prior art suggesting that specific chemokines such as RANTES, MIP-1xcex1 and MIP-1xcex2 may inhibit fusion between the HIV-1 virion and target cells by inhibiting the interaction between HIV surface proteins and cell surface receptors. This inhibits viral replication by reducing the rate of infection.
Fusion between the HIV virion and the plasma membrane of the target cell allows the HIV RNA to enter the target cell, where it is reverse transcribed into DNA by viral reverse transcriptase and integrated into the host cell""s genome to form an HIV provirus. Once the HIV DNA is integrated into the host cell genome, it is replicated during cell division and is passed on to daughter cells.
The HIV provirus may remain inactive in the host cell for some time until it is activated. Upon activation, HIV structural genes are expressed, and single stranded HIV RNA (HIV ssRNA) is transcribed. The HIV structural proteins and HIV ssRNA assemble to form numerous virus particles which then exits from the host cell infects other cells.
At present, methods of inhibiting HIV replication in tissue samples have tended to focus on reducing the number of newly infected cells through the inhibition of infection by released virus particles. This has been effected through the use of compounds which inhibit fusion between the HIV virion and the plasma membrane, and inhibitors of viral reverse transcriptase (necessary to generate DNA from the viral RNA prior to integration into the host cell genome) to form the provirus. In addition, the production and release of viral particles from infected cells has been inhibited through the use of protease inhibitors which interfere with the post-translational processing of HIV gene products necessary for virus particle formation. The effectiveness of many current therapies is limited by the capacity of the HIV virus to mutate, resulting in the development of resistance. Methods for inhibiting the expression of HIV DNA in populations comprising CD8 and CD4 cells from infected subjects (thereby reducing the number of virus particles which can be formed) while greatly expanding CD4 and CD8 cells in these populations are not known in the art. Such methods might be less susceptible to circumvention by acquired resistance and therefore represent a potentially powerful form of HIV treatment.
It is desirable to have a means of inhibiting the expression of HIV DNA in infected cells. Individual infected cells are capable of producing a massive number of infectious HIV particles, and the release of such particles from a cell can cause the infection of numerous previously uninfected cells Levine et al. (Science, 272:1939, Jun. 18, 1996) have reported that the interaction of CD4 cells with immobilized (but not soluble) CD28 monoclonal antibodies reduces the susceptibility of CD4 cells to HIV infection. However, it would be more efficient to inhibit the formation of HIV particles in infected cells, rather than to simply attempt to reduce the rate of infection by such particles following their formation and release from the infected cell.
HIV DNA sequences are flanked by long terminal repeats (LTRs). Promoter and enhancer sequences are located in the 5xe2x80x2 LTR, and polyadenylation sequences are contained in the 3xe2x80x2 LTR. The 5xe2x80x2 LTR sequence normally has only a low affinity for RNA polymerase, causing premature truncation of transcription products and preventing the formation of infectious viral particles. However, the viral protein Tat is capable of interacting specifically with a region (TAR) on the emerging RNA transcript and increasing the formation of full-length proviral transcripts.
One of the characteristic features of HIV infection is a reduction in the number of CD4+ T-cells (xe2x80x9cCD4 cellsxe2x80x9d) in the peripheral blood of infected subjects. Healthy uninfected individuals typically have approximately 1100 CD4 cells per microliter of whole blood. After an individual has been infected with HIV, CD4 cell levels generally drop gradually over a period of 8 to 10 years, but then drops more rapidly. In subjects with AIDS, CD4 cells levels below 200 cells per xcexcl are common.
It is believed that in the early stages of HIV infection CD4 cells are destroyed at a high rate. However, at this stage the subject""s immune system is able to replace many of the destroyed cells, resulting in only a gradual decline in observed CD4 cell numbers. Cells may be destroyed by various means following infection. One means for the destruction of infected cells is lysis resulting from the exit of large numbers of newly formed virs particles. A second means by which infected cells may be destroyed is by an immune response to HIV antigens expressed on the cell membrane. It is believed that enhanced levels of active anti-HIV specific CD4 cells in HIV infected patients allows the maintenance of low viral loads and non-progression into AIDS.
It appears that many uninfected CD4 cells lose their capacity to respond to foreign antigens and are also destroyed during HIV infection. The exact mechanism by which this occurs is not fully understood. However, it is suspected that free gp120 to CD4 molecules on the surface of uninfected cells. This binding may lead to the internalization of the gp120 by the uninfected CD4 cells. Proteolytic processing of the internalized gp120 in the endosome, followed by association of the processed peptides with class II MHC, may lead to the expression of an HIV peptide-MHC complex at the surface of uninfected cells. Such cells may thus be destroyed as a result of an immune response directed at the peptide-MHC complex. Additionally, the binding of free gp120 to CD4 molecules on uninfected cells may interfere with the ability of these CD4 molecules to interact with class II MHC molecules on antigen-presenting cells, thereby reducing the ability of the uninfected cell to participate in an immune response to foreign antigen. Alternately, the binding of gp120 to CD4 molecules on an uninfected CD4 cell may stimulate the production of an inappropriate activation signal, which may lead to apoptosis. It has also been postulated the free gp120 may bind to CD4 molecules on developing thymocytes, interfering with normal T cell maturation processes. Additionally, there is evidence suggesting that a single CD4 cell infected with HIV can fuse with large numbers of uninfected CD4 cells, forming a syncytium. Syncytia appear capable of producing large numbers of viral particles over a short period of time before they die.
There is evidence indicating that CD4 cell populations from subjects with AIDS have a significantly reduced ability to proliferate in response to specific antigens. This selective loss of responsiveness has been hypothesized to be the result of an inappropriate activating signal received by CD4cells, leading to cellular anergy or apoptosis.
A shift in cytokine production by CD4 cells has been observed during the progression toward AIDS. As the disease progresses, the production of the Th1-type cytokines IL-2 and IFN-xcex3 decreases and the production of the Th2 type cytokine IL-10 (and for a limited time IL-4) increases. This may reflect a shift from a Th1-type cellular immune response to a Th2-type humoral immune response. The reduction in IL-2 levels observed following HIV infection appears to impair the ability of CD8 cells to form cytotoxic T-lymnphocytes, reducing the subject""s ability to eliminate virus-infected cells and tumour cells. IFNxcex3 has been reported to induce an anti-viral state in cells, and reduced IFNxcex3 levels following HIV infection may undermine this mechanism of cellular defense. Furthermore, IL-2 and IFNxcex3 activate natural killer cells (xe2x80x9cNK cellsxe2x80x9d) which are important in the very early stages of viral infection.
The cellular depletion observed following HIV infection appears to primarily affect the CD4 cell population. However, the infection and eventual loss of dendritic cells may play an important role in disease progression. Dendritic cells are major antigen presenting cells and are important to T cell activation. Dendritic cells are also important in the maintenance of functional lymph nodes, wherein T cell and B cell activation occurs.
It has been estimated that subjects infected with HIV, but not yet diagnosed as having AIDS, lose approximately two billion CD4 cells each day. While some of these cells will be replaced by the subject""s own immune system, cell numbers eventually decline. Additionally, free gp120 may interact with uninfected CD4 cells of HIV-infected subjects, thereby reducing the effectiveness of surviving CD4 cells. It has therefore been proposed to produce large populations of CD4 cells for transfusion into HIV-infected subjects to replace cells destroyed or inactivated due to infection.
CD8 cell levels in the blood of HIV infected subjects are typically near normal. However, cytotoxic T lymphocyte (xe2x80x9cCTLxe2x80x9d) activity is generally impaired in AIDS patients. Cell mediated immune responses are the principle immunological defense to HIV infection and a vigorous CTL response early in infection has been associated with a lower rate of disease progression. CTL""s are the major effector cells in this antiviral response. However, the decline in CTL activity observed following HIV infection suggests that anti-HIV activity by CTL""s is impaired. Thus, it desirable to have a means to induce the formation and/or proliferation of CTL""s, and, even more preferably, the formation of HIV-specific CTL""s. It would also be highly desirable to have a means of expanding CD4 cells, CD8 cells and CD4+ CD8+ T-cells (xe2x80x9cDP cellsxe2x80x9d) from HIV infected subjects, particularly if this could be accomplished while keeping viral levels in the cultured cells low. DP cells represent an early stage in T cell development and can mature to form CD4 cells or CD8 cells having varying antigenic specificities.
In order to maximize the effectiveness of treatment with expanded T cells, it is desirable that the infused cells be MHC compatible with the subject""s tissue. Ideally, the best MHC match will be obtained by using the subject""s own cells for infusion. However, it would be inadvisable to remove large numbers of cells from the blood of an HIV-infected patient for culture or expansion, as this may further compromise the subject""s ability to mount effective immune responses against foreign antigens.
It is an object of the invention to provide a method to expand a population of T cells from HIV-infected subjects which will allow the production of enough cells to be effective in bolstering the subject""s immune response from an initial sample which is small enough so that its removal does not pose a significant health risk to the subject.
It is a further method of the invention to provide a method of expanding a cell population contain HIV-infected cells which does not lead to the production of high levels of HIV in the expanded population.
It is a further object of the invention to provide a method to interfere with the expression of HIV DNA in the expanded cell population.
It is a further object of the invention to provide a use of CM to inhibit the expression of HIV DNA in a cell infected with an HIV provirus.
It is a further object of the invention to provide a method of screening cell populations derived from HIV infected subjects for susceptibility to one or more anti-HIV treatments.
It is a further object of the invention to provide a use for CM in screening cells from HIV infected subjects for susceptibility to one or more anti-HIV treatments.
It is a further object of the invention to provide a use for CM to generate cell banks.
It is a further object of the invention to provide a composition of matter comprising an expanded population of CD8 cells derived from an HIV infected patient.
It is a further object of the invention to provide a composition of matter comprising an expanded population of DP cells derived from an HIV infected patient.
It is a further object of the invention to provide a use of CM to obtain a late culture cell population from a T cell sample obtained from an HIV infected subject.
It is a further object of the invention to provide a composition of matter comprising a substantially pure late culture population derived from a T cell population obtained from an HIV infected subject.